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The protein phosphatase 5 (PP5) is normally recruited to its substrates by the molecular chaperones, heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90). This interaction requires the tetratricopeptide repeat (TPR) domain of PP5, which binds to an EEVD motif at the extreme C termini of cytosolic Hsp70 and Hsp90 isoforms. In addition to bringing PP5 into proximity with chaperone-bound substrates, this interaction also relieves autoinhibition in PP5s catalytic domain, promoting its phosphatase activity. To better understand the molecular determinants of this process, we screened a large, pentapeptide library for binding to PP5. This screen identified the amino acid preferences at each position, which we validated by showing that the optimal sequences bind 4- to 7-fold tighter than the natural EEVD motifs and stimulate PP5s enzymatic activity. The enhanced affinity for PP5s TPR domain was confirmed using a protein-adaptive differential scanning fluorimetry assay. Using this increased knowledge of structure-activity relationships, we re-examined affinity proteomics results to look for potential EEVD-like motifs in the C termini of known PP5-binding partners. This search identified elongator acetyltransferase complex subunit 1 (IKBKAP) as a putative partner, and indeed, we found that its C-terminal sequence, LSLLD, binds directly to PP5s TPR domain in vitro. Consistent with this idea, mutation of elongator acetyltransferase complex subunit 1s terminal aspartate was sufficient to interrupt the interaction with PP5 in vitro and in cells. Together, these findings reveal the sequence preferences of PP5s TPR domain and expand the scope of PP5s functions to include chaperone-independent complexes.

High pressure processing (HPP) offers the benefits of safety, uniformity, energy-efficient, and low waste, which is widely applied for microbial inactivation and shelf-life extension for foods. Over the past forty years, HPP has been extensively researched in the food industry, enabling the inactivation or activation of different enzymes in future food by altering their molecular structure and active site conformation. Such activation or inactivation of enzymes effectively hinders the spoilage of food and the production of beneficial substances, which is crucial for improving food quality. This paper reviews the mechanism in which high pressure affects the stability and activity of enzymes, concludes the roles of key enzymes in the future food processed using high pressure technologies. Moreover, we discuss the application of modified enzymes based on high pressure, providing insights into the future direction of enzyme evolution under complex food processing conditions (e.g. high temperature, high pressure, high shear, and multiple elements). Finally, we conclude with prospects of high pressure technology and research directions in the future. Although HPP has shown positive effects in improving the future food quality, there is still a pressing need to develop new and effective combined processing methods, upgrade processing modes, and promote sustainable lifestyles. [ABSTRACT FROM AUTHOR]

Background: Milk clotting enzymes, essential for milk coagulation in cheese production, are obtained from the stomach of young ruminants, an expensive and limited source. This study was accomplished by finding a suitable alternative. Bacterial isolates recovered from honey were screened for milk clotting enzyme activity. and further, by immobilization of the microorganisms to enhance stability and facilitate their repeated use. Result: The most effective enzyme was produced by a microbe identified as Bacillus amyloliquefaciens based on 16 S rRNA sequencing. The cells were encapsulated in Ca2+ alginate beads. These beads retained complete enzyme production after being used five times. Glucose and Soybean were selected as the most favorable carbon and nitrogen sources, respectively. The optimum temperature for activity was 35 ℃ for both free and immobilized cells but as the temperature was increased to 55 °C and above, the encapsulated form retained more activity than the free cells. The pH optimum shifted from 6.5 to 7 for the free cells to 7–7.5 for the immobilized cells. The immobilization process decreased the activation energy for enzyme production and activity, prolonged the enzyme half-life, and increased the deactivation energy. Enzyme produced by immobilized cells generated a more compact cheese. Conclusions: The finding of this study was to identify a less expensive source of milk-clotting enzymes and confirm the success of cell immobilization in improving cell rigidity and stability. Also, immobilization of this B. amyloliquefaciens strain offers an enzyme source of value for industrial production of cheese. [ABSTRACT FROM AUTHOR]

Pyruvate:quinone oxidoreductase (PQO) is a flavin‐containing peripheral membrane enzyme catalyzing the decarboxylation of pyruvate to acetate and CO2 with quinone as an electron acceptor. Here, we investigate PQO activity in Corynebacterium glutamicum, examine purified PQO, and describe the crystal structure of the native enzyme and a truncated version. The specific PQO activity was highest in stationary phase cells grown in complex medium, lower in cells grown in complex medium containing glucose or acetate, and lowest in cells grown in minimal acetate‐medium. A similar pattern with about 30‐fold higher specific PQO activities was observed in C. glutamicum with plasmid‐bound pqo expression under the control of the tac promoter, indicating that the differences in PQO activity are likely due to post‐transcriptional control. Continuous cultivation of C. glutamicum at dilution rates between 0.05 and 0.4 h−1 revealed a negative correlation between PQO activity and growth rate. Kinetic analysis of PQO enzymes purified from cells grown in complex or in minimal acetate‐medium revealed substantial differences in specific activity (72.3 vs. 11.9 U·mg protein−1) and turnover number (kcat: 440 vs. 78 s−1, respectively), suggesting post‐translational modifications affecting PQO activity. Structural analysis of PQO revealed a homotetrameric arrangement very similar to the Escherichia coli pyruvate oxidase PoxB except for the C‐terminal membrane binding domain, which exhibited a conformation markedly different from its PoxB counterpart. A truncated PQO variant lacking 17 C‐terminal amino acids showed higher affinity to pyruvate and was independent of detergent activation, highlighting the importance of the C‐terminus for enzyme activation and lipid binding. [ABSTRACT FROM AUTHOR]

Soaking and germination are processes used to improve nutritional characteristics in rice especially concerning γ‐aminobutyric acid (GABA) and phenolic compounds. In contrast, during these processes the activation of enzymes may affect the rice starch. Hence, the current investigation aims to understand the impact of soaking and germination processes on starch structure in terms of morphological, texture, physicochemical and gelatinization properties of non‐germinated rice starch (NGRS), soaked rice starch (SRS), and germinated rice starch (GRS). It is observed that soaking and germination cause changes in granule surface and reduce relative crystallinity (27.40–26.08 and 27.40–25.65%) and enthalpy (9.69–8.81 and 9.69–8.37%) compared with NGRS. Unexpectedly, germination affects the gel strength during cooling showing an increase of maximum peak force (0.28 N), demonstrating that the gel of GRS is firmer than NGRS and SRS. The findings are useful to provide bases for utilization of soaked and germinated rice starches in developing products for the food industry. [ABSTRACT FROM AUTHOR]

Receptor guanylyl cyclases (GCs) are single membrane spanning, multidomain enzymes, that synthesize cGMP in response to natriuretic peptides or other ligands. They are evolutionarily conserved from sea urchins to humans and regulate diverse physiologies. Most family members are phosphorylated on 4 to 7 conserved serines or threonines at the beginning of their kinase homology domains. This review describes studies that demonstrate that phosphorylation and dephosphorylation are required for activation and inactivation of these enzymes, respectively. Phosphorylation sites in GC-A, GC-B, GC-E, and sea urchin receptors are discussed, as are mutant receptors that mimic the dephosphorylated inactive or phosphorylated active forms of GC-A and GC-B, respectively. A salt bridge model is described that explains why phosphorylation is required for enzyme activation. Potential kinases, phosphatases, and ATP regulation of GC receptors are also discussed. Critically, knock-in mice with glutamate substitutions for receptor phosphorylation sites are described. The inability of opposing signaling pathways to inhibit cGMP synthesis in mice where GC-A or GC-B cannot be dephosphorylated demonstrates the necessity of receptor dephosphorylation in vivo. Cardiac hypertrophy, oocyte meiosis, long-bone growth/achondroplasia, and bone density are regulated by GC phosphorylation, but additional processes are likely to be identified in the future. [ABSTRACT FROM AUTHOR]

Dynamic constitutional materials are constructed from reversibly connecting components adaptively self-assembling under the pressure of internal or external factors. Functional macromonomeric poly-(ethylene glycol) segments and 3D connecting cores can be used to conceive biomimetic dynamic constitutional frameworks (DCFs) for selective/adaptive biomolecular encapsulation of proteins, enzymes, etc. Gold nanoparticles (AuNps) can be involved as core connectors to expand the networks of DCFs at the nanoscale, thereby increasing the biomolecular encapsulation and activity. Efficient encapsulation of bovine carbonic anhydrase (bCA) in host matrixes is of tremendous importance for maintaining its stability, catalytic activity, and durability. In this paper, we demonstrate that AuNps-DCFs-bCA bioconjugates, synthesized via reversible imine/amino-carbonyl as sulfide-AuNps chemistries, assemble into nanometric aggregates of around 200 nm with high stability for bCA encapsulation. This system showed an impressive improved catalytic activity, more than 30 times higher than that of bCA alone. The Km values were two to three times lower compared to free bCA indicating a stronger affinity for bCA under encapsulation, while Vmax was higher. This performance is very promising for current state-of-the-art carbon dioxide capture systems with equivalent amounts of enzyme, even after heating for a prolonged period at 100 °C, translating into a direct perspective for enhancing flue gas capture and conversion performances. [ABSTRACT FROM AUTHOR]

Introduction: Pulmonary embolism (PE), the most serious presentation of venous thromboembolism (VTE), is associated with a high rate of mortality and expense. Clinical studies on pregnant women with PE are scarce. The aim of this study was to analyze the clinical impact of fibrinolytic enzyme activation inhibitor-1 (PAI-1) 4G/5G genetic polymorphisms, methylenetetrahydrofolate reductase (MTHFR) rs1801131 (A1298C) and rs1801133 (C677T) genetic polymorphisms, and establish a predictive model for pregnant women. Material and Methods: Between September 2022 and August 2023, 53 pregnant women with PE were enrolled. Using the propensity score matching method, 106 consecutive pregnant women without VTE were 1:2 matched. The relevant patient data were collected, and the susceptibility genes for PE were detected to determine genetic polymorphisms, and PE susceptibility in pregnant women, as well as to develop predictive models. Results: Our study showed that 4G/4G homozygous mutations increased the risk of pregnant PE fourfold (OR = 4.46, 95% CI = 1.59– 12.50, P = 0.004), whereas the 4G allele mutation increased the risk twofold (OR = 2.33, 95% CI = 1.35– 4.04, P = 0.002). A nomogram was established to predict the risk of pregnant women with PE by four predictive features including PAI-1 genetic polymorphisms, international normalized ratio (INR), antithrombin-III (AT-III) activity, and platelet count (PLT). The area under the curve (AUC) of the nomogram was 0.821 (0.744– 0.898). The AUC of the internal validation group was 0.822 (0.674– 0.971). Decision curve analysis revealed that the nomogram has a higher net benefit in the following threshold: probability interval of ≥ 15%. Conclusion: The PAI-1 4G/4G genotype is an independent risk factor for pregnant women with PE; furthermore, the presence of the 4G allele can increase the risk of PE. The study established a nomogram to predict the risk of PE in pregnant women. [ABSTRACT FROM AUTHOR]

The Eastern Long-Nosed Viper (Vipera ammodytes meridionalis) is considered one of the most venomous snakes in Europe. However, it is unknown whether ontogenetic variation in venom effects occurs in this subspecies and how this may impact antivenom efficacy. In this study, we compared the procoagulant activities of V. a. meridionalis venom on human plasma between neonate and adult venom phenotypes. We also examined the efficacy of three antivenoms—Viperfav, ViperaTAb, and Inoserp Europe—across our neonate and adult venom samples. While both neonate and adult V. a. meridionalis venoms produced procoagulant effects, the effects produced by neonate venom were more potent. Consistent with this, neonate venom was a stronger activator of blood-clotting zymogens, converting them into their active forms, with a rank order of Factor X >> Factor VII > Factor XII. Conversely, the less potent adult venom had a rank order of FXII marginally more activated than Factor VII, and both much more so than Factor X. This adds to the growing body of evidence that activation of factors besides FII (prothrombin) and FX are significant variables in reptile venom-induced coagulopathy. Although all three examined antivenoms displayed effective neutralization of both neonate and adult V. a. meridionalis venoms, they generally showed higher efficacy on adult venom than on neonate venom. The ranking of antivenom efficacy against neonate venom, from the most effective to the least effective, were Viperfav, Inoserp Europe, ViperaTAb; for adult venom, the ranking was Inoserp Europe, Viperfav, ViperaTAb. Our data reveal ontogenetic variation in V. a meridionalis, but this difference may not be of clinical concern as antivenom was effective at neutralizing both adult and neonate venom phenotypes. Regardless, our results highlight a previously undocumented ontogenetic shift, likely driven by the documented difference in prey preference observed for this species across age classes [ABSTRACT FROM AUTHOR]

Pulsed light is an emerging technique in plant physiology recognized for its ability to enhance germination and accumulate γ–aminobutyric acid in maize. Pulsed light involves exposing plants to brief, high-intensity bursts of light, which can enhance photosynthesis, improve growth, and increase resistance to environmental stresses. Despite its promising potential, the specific metabolic changes leading to γ–aminobutyric acid enrichment in maize induced by pulsed light are not fully understood. This study addresses this gap by quantifying key nutrients and γ–aminobutyric acid-related compounds during maize germination and investigating the underlying mechanisms using non-targeted metabolomics. Our findings indicate that pulsed light significantly promotes maize germination and accelerates the hydrolysis of proteins, sugars, and lipids. This acceleration is likely due to the activation of enzymes involved in these metabolic pathways. Additionally, pulsed light markedly increases the content of glutamic acid and the activity of glutamate decarboxylase, which are crucial for γ–aminobutyric acid synthesis. Moreover, pulsed light significantly reduces the activity of γ–aminobutyric transaminase, thereby inhibiting γ–aminobutyric acid decomposition and resulting in a substantial increase in γ–aminobutyric acid content, with a 27.20% increase observed in germinated maize following pulsed light treatment. Metabolomic analysis further revealed enrichment of metabolic pathways associated with γ–aminobutyric acid, including amino acid metabolism, carbohydrate metabolism, plant hormone signal transduction, energy metabolism, pyrimidine metabolism, and ABC transporters. In conclusion, pulsed light is a robust and efficient method for producing sprouted maize with a high γ–aminobutyric acid content. This technique provides a novel approach for developing sprouted cereal foods with enhanced nutritional profiles, leveraging the physiological benefits of γ–aminobutyric acid, which include stress alleviation and potential health benefits for humans. [ABSTRACT FROM AUTHOR]

Macrophages represent primary players of the innate immune system. Macrophage activation triggers several signaling pathways and is tightly associated with metabolic changes, which drive different immune subsets. Recent studies unveil the role of various metabolic enzymes in macrophage activation. Here, we show that malic enzyme 1 (ME1) is overexpressed in LPS-induced macrophages. Through chromatin immunoprecipitation, we demonstrate that ME1 transcriptional regulation is under control of NF-κB. Furthermore, ME1 activity is also increased in activated human PBMC-derived macrophages. Notably, ME1 gene silencing decreases nitric oxide as well as reactive oxygen species and prostaglandin E2 inflammatory mediators. Therefore, modulating ME1 provides a potential approach for immunometabolic regulation and in turn macrophage function. [ABSTRACT FROM AUTHOR]

Bemnifosbuvir (AT-527) and AT-752 are guanosine analogues currently in clinical trials against several RNA viruses. Here, we show that these drugs require a minimal set of 5 cellular enzymes for activation to their common 5′-triphosphate AT-9010, with an obligate order of reactions. AT-9010 selectively inhibits essential viral enzymes, accounting for antiviral potency. Functional and structural data at atomic resolution decipher N6-purine deamination compatible with its metabolic activation. Crystal structures of human histidine triad nucleotide binding protein 1, adenosine deaminase-like protein 1, guanylate kinase 1, and nucleoside diphosphate kinase at 2.09, 2.44, 1.76, and 1.9 Å resolution, respectively, with cognate precursors of AT-9010 illuminate the activation pathway from the orally available bemnifosbuvir to AT-9010, pointing to key drug–protein contacts along the activation pathway. Our work provides a framework to integrate the design of antiviral nucleotide analogues, confronting requirements and constraints associated with activation enzymes along the 5′-triphosphate assembly line. Bemnifosbuvir (AT-527) and AT-752 are broad-spectrum antiviral guanosine analogues currently in clinical trials. This study reconstitutes their intracellular 5-step enzymatic activation pathway, characterizing each enzymatic complex at atomic resolution, which provides insight into substrate binding and drug activation. [ABSTRACT FROM AUTHOR]

Studies on the immune-regulatory roles played by the commensal microbes residing in the nasal mucosa consider the contribution of antiviral immune responses. Here, we sought to identify the nasal microbiome, Staphylococcus epidermidis-regulated antiviral immune responses and the alteration of polyamine metabolites in nasal epithelium. We found that polyamines were required for the life cycle of influenza A virus (IAV) and depletion of polyamines disturbed IAV replication in normal human nasal epithelial (NHNE) cells. Inoculation of S. epidermidis also suppressed IAV infection and the concentration of polyamines including putrescine, spermidine, and spermine was completely attenuated in S. epidermidis-inoculated NHNE cells. S. epidermidis activated the enzyme involved in the production of ornithine from arginine and downregulated the activity of the enzyme involved in the production of putrescine from ornithine in nasal epithelium. S. epidermidis also induced the activation of enzymes that promote the extracellular export of spermine and spermidine in NHNE cells. Our findings demonstrate that S. epidermidis is shown to be able of creating an intracellular environment lacking polyamines in the nasal epithelium and promote the balance of cellular polyamines in favor of the host to restrict influenza virus replication. Staphylococcus epidermidis, a nasal commensal in healthy mucus, can create an intracellular epithelium environment lacking polyamines and promote balance of cellular polyamines in favor of the host to restrict influenza virus replication. [ABSTRACT FROM AUTHOR]

Germination is an effective measure to regulate the accumulation of secondary metabolites in plants. In this study, we optimized the germination conditions of finger millet by response surface methodology. Meanwhile, physiological characteristics and gene expression were measured to investigate the mechanism of flavonoid accumulation in finger millet at the germination stage. The results showed that when germination time was 5.7 d, germination temperature was 31.2 °C, and light duration was 17.5 h, the flavonoid content of millet sprouts was the highest (7.0 μg/sprout). The activities and relative gene expression of key enzymes for flavonoid synthesis (phenylalanine ammonia-lyase, 4-coumarate-coenzyme a ligase, and cinnamate 4-hydroxylase) were significantly higher in finger millet sprouts germinated at 3 and 5 d compared with that in ungerminated seeds (p < 0.05). In addition, germination enhanced the activities of four antioxidant enzymes (catalase, peroxidase, superoxide dismutase, and ascorbate peroxidase) and up-regulated the gene expression of PAL and APX. Germination increased malondialdehyde content in sprouts, which resulted in cell damage. Subsequently, the antioxidant capacity of the sprouts was enhanced through the activation of antioxidant enzymes and the up-regulation of their gene expression, as well as the synthesis of active substances, including flavonoids, total phenolics, and anthocyanins. This process served to alleviate germination-induced cellular injury. These findings provide a research basis for the regulation of finger millet germination and the enhancement of its nutritional and functional properties. [ABSTRACT FROM AUTHOR]

Phosphoryl transfer is a fundamental reaction in cellular signaling and metabolism that requires Mg2+ as an essential cofactor. While the primary function of Mg2+ is electrostatic activation of substrates, such as ATP, the full spectrum of catalytic mechanisms exerted by Mg2+ is not known. In this study, we integrate structural biology methods, molecular dynamic (MD) simulations, phylogeny, and enzymology assays to provide molecular insights into Mg2+-dependent structural reorganization in the active site of the metabolic enzyme adenylate kinase. Our results demonstrate that Mg2+ induces a conformational rearrangement of the substrates (ATP and ADP), resulting in a 30° adjustment of the angle essential for reversible phosphoryl transfer, thereby optimizing it for catalysis. MD simulations revealed transitions between conformational substates that link the fluctuation of the angle to large-scale enzyme dynamics. The findings contribute detailed insight into Mg2+ activation of enzymes and may be relevant for reversible and irreversible phosphoryl transfer reactions. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus signal peptidase IB (SpsB) is an essential enzyme for protein secretion. While inhibition of its activity by small molecules is a well-precedented mechanism to kill bacteria, the mode of activation is however less understood. We here investigate the activation mechanism of a recently introduced activator, the antibiotic compound PK150, and demonstrate by combined experimental and Molecular Dynamics (MD) simulation studies a unique principle of enzyme stimulation. Mass spectrometric studies with an affinity-based probe of PK150 unravel the binding site of PK150 in SpsB which is used as a starting point for MD simulations. Our model shows the localization of the molecule in an allosteric pocket next to the active site which shields the catalytic dyad from excess water that destabilizes the catalytic geometry. This mechanism is validated by the placement of mutations aligning the binding pocket of PK150. While the mutants retain turnover of the SpsB substrate, no stimulation of activity is observed upon PK150 addition. Overall, our study elucidates a previously little investigated mechanism of enzyme activation and serves as a starting point for the development of future enzyme activators. The study explores the activation mechanism of Staphylococcus aureus signal peptidase IB by the antibiotic PK150. We identified that PK150 shields the active site from excess water and stabilizes its catalytic geometry, thus enhancing catalysis. [ABSTRACT FROM AUTHOR]

We report on the synthesis of two fluorescent probes which can be activated by β-Galactosidase (β-Gal) enzymes and/or light. The probes contained 2-nitro-4-oxybenzyl and 3-nitro-4-oxybenzyl fragments, with β-Gal residues linked to C-4. We performed the enzymatic and photoactivation of the probes in a cuvette and compared them, prior to the labeling of Vimentin–Halo fusion protein in live cells with overexpressed β-galactosidase. The dye fluorescence afforded the observation of enzyme activity by means of confocal and super-resolution optical microscopy based on stimulated emission depletion (STED). The tracing of enzymatic activity with the retention of activated fluorescent products inside cells was combined with super-resolution imaging as a tool for use in biomedicine and life science. [ABSTRACT FROM AUTHOR]

This review discusses the potential of targeting the kynurenine pathway (KP) in the treatment of inflammatory diseases. The KP, responsible for the catabolism of the amino acid tryptophan (TRP), produces metabolites that regulate various physiological processes, including inflammation, cell cycle, and neurotransmission. These metabolites, although necessary to maintain immune balance, may accumulate excessively during inflammation, leading to systemic disorders. Key KP enzymes such as indoleamine 2,3-dioxygenase 1 (IDO1), indoleamine 2,3-dioxygenase 2 (IDO2), tryptophan 2,3-dioxygenase (TDO), and kynurenine 3-monooxygenase (KMO) have been considered promising therapeutic targets. It was highlighted that both inhibition and activation of these enzymes may be beneficial, depending on the specific inflammatory disorder. Several inflammatory conditions, including autoimmune diseases, for which modulation of KP activity holds therapeutic promise, have been described in detail. Preclinical studies suggest that this modulation may be an effective treatment strategy for diseases for which treatment options are currently limited. Taken together, this review highlights the importance of further research on the clinical application of KP enzyme modulation in the development of new therapeutic strategies for inflammatory diseases. [ABSTRACT FROM AUTHOR]

Linum album is a rich source of various phenolic compounds, especially lignans. Our previous studies revealed that phenylalanine (Phe), an aromatic amino acid, participates in lignans production. In this study, we tried to characterize how Phe as the precursor feeding affects metabolic fluxes in L. album cells. As expected, the results indicated that an increase in phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase activities (TAL) occurred at 250 μM. Their actions induced phenolic acids production, especially cinnamic acid and salicylic acid (SA). The low dosages of Phe stimulated hydrogen peroxide (H2O2) and nitric oxide (NO) generation, lipid peroxidation, and antioxidant enzymes activation, while the high dosages suppressed the mentioned physiological processes. A depletion in rhamnose/xylose, glucose, and mannose was observed at high concentrations of Phe compared with the control. These molecules altered amino acids profile through supplying carbon skeleton and cell required energy. Our observations also showed that amino acids contents were significantly influenced by Phe treatment. The reduction of endogenous Phe content in the treated cells may closely related to phenolics accumulation such as flavonoids and lignans, as the enhancement of catechin, myricetin, and kaempferol were observed in the treated cells. Likewise, phenylalanine increased lignans, except for lariciresionol (LARI), and the highest amounts of podophyllotoxin (PTOX), and 6-methoxy podophyllotoxin (6MPTOX) were detected at 1000 μM. In summary, metabolic and physiological changes can elucidate that Phe involves in the lignans production in L. album through a SA-dependent pathway. [ABSTRACT FROM AUTHOR]

Acute pancreatitis (AP) is a sudden-onset disease of the digestive system caused by abnormal activation of pancreatic enzymes. Dual oxidase 2 (DUOX2) has been found to be elevated in the progression of a variety of inflammatory diseases. Therefore, we analyzed the specific roles of DUOX2 in AP development. Blood samples were collected from of AP patients and healthy people, and the caerulein- stimulated human pancreatic duct cells (H6C7) were utilized to establish an AP cell model. Cell growth and apoptosis were measured using an MTT assay and TUNEL staining. Additionally, RT-qPCR and western blot assays were conducted to assess the RNA and protein expressions of the cells. ELISA kits were used to determine TNF-α, IL-6, IL-8, and IL-1β levels. The interaction between DUOX2 and miR-605-3p was predicted using the Targetscan database and confirmed by dual-luciferase report assay. We found that DUOX2 increased while miR-605-3p decreased in the blood of AP patients and caerulein-stimulated H6C7 cells. DUOX2 was targeted by miR-605-3p. Furthermore, DUOX2 knockdown or miR-605-3p overexpression promoted cell viability, decreased the TNF-α, IL-6, IL-8, and IL-1β levels, and inhibited apoptosis rate in caerulein-stimulated H6C7 cells. DUOX2 knockdown or miR-605-3p overexpression also increased the Bcl-2 protein levels and down-regulated Bax, cleaved-caspase-1, NLRP3 and p-p65. Interestingly, DUOX2 overexpression reversed the miR-605-3p mimic function in the caerulein-treated H6C7 cells. In conclusion, our research demonstrated that DUOX2 knockdown relieved the injury and inflammation in caerulein-stimulated H6C7 cells. [ABSTRACT FROM AUTHOR]

The Sogatella furcifera (Horva'th) (Homoptera: Delphacidae) is a white-backed planthopper (WBPH) that causes "hopper burn" in rice, resulting in severe yield loss. Gamma-aminobutyric acid (GABA) is a well-known neurotransmitter that inhibits neurotransmission in insects by binding to specific receptors. In this study, we investigated the potential role of GABA in modulating rice resistance to WBPH and evaluated possible defense mechanisms. The experiment was conducted in green house in pots consist of four groups: control, GABAtreated, WBPH-infested, and WBPH-infested treated with GABA. Among the various tested concentration of GABA, 15 mM GABA was applied as a single treatment in water. The treatment was administered one week before WBPH infestation. The results revealed that 15 mM GABA treatment strongly increased WBPH resistance. A plate-based assay indicated that direct application of 15 mM GABA increased the mortality rate of WBPH and increased the damage recovery rate in rice plants. We found that GABA treatment increased the activation of antioxidant enzymes and reduced the reactive oxygen species content and malondialdehyde contents, and reduced the damage rate caused by WBPH. Interestingly, GABA-supplemented plants infested with WBPH exhibited increased phenylalanine ammonia-lyase and pathogenesis-related (PR) genes expression levels. GABA induced the accumulation of abscisic acid (ABA) and salicylic acid (SA) and enhanced the stomata closure and reduced leaf vessels to reduce water conductance during WBPH stress. Furthermore, we found that GABA application to the plant induced the expression of Jasmonic acid (JA) biosynthesis genes (LOX, AOS, AOC, and OPR) and melatonin biosynthesisrelated genes (TDC, T5H, ASMT, and SNAT). Our study suggested that GABA increases resistance against WBPH infestation by regulating antioxidant defense system, TCA cycle regulation, phytohormonal signaling, and PR gene regulation. [ABSTRACT FROM AUTHOR]

β-arrestins play a key role in G protein-coupled receptor (GPCR) internalization, trafficking, and signaling. Whether β-arrestins act independently of G protein-mediated signaling has not been fully elucidated. Studies using genome-editing approaches revealed that whereas G proteins are essential for mitogen-activated protein kinase activation by GPCRs., β-arrestins play a more prominent role in signal compartmentalization. However, in the absence of G proteins, GPCRs may not activate β-arrestins, thereby limiting the ability to distinguish G protein from β-arrestin-mediated signaling events. We used β2-adrenergic receptor (β2AR) and its β2AR-C tail mutant expressed in human embryonic kidney 293 cells wildtype or CRISPR-Cas9 gene edited for Gαs, β-arrestin1/2, or GPCR kinases 2/3/5/6 in combination with arrestin conformational sensors to elucidate the interplay between Gαs and β-arrestins in controlling gene expression. We found that Gαs is not required for β2AR and β-arrestin conformational changes, β-arrestin recruitment, and receptor internalization, but that Gαs dictates the GPCR kinase isoforms involved in β-arrestin recruitment. By RNA-Seq analysis, we found that protein kinase A and mitogen-activated protein kinase gene signatures were activated by stimulation of β2AR in wildtype and β-arrestin1/2-KO cells but absent in Gαs-KO cells. These results were validated by re-expressing Gαs in the corresponding KO cells and silencing β-arrestins in wildtype cells. These findings were extended to cellular systems expressing endogenous levels of β2AR. Overall, our results support that Gs is essential for β2AR-promoted protein kinase A and mitogen-activated protein kinase gene expression signatures, whereas β-arrestins initiate signaling events modulating Gαs-driven nuclear transcriptional activity.

Mutations in the tyrosine phosphatase Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) are associated with a variety of human diseases. Most mutations in SHP2 increase its basal catalytic activity by disrupting autoinhibitory interactions between its phosphatase domain and N-terminal SH2 (phosphotyrosine recognition) domain. By contrast, some disease-associated mutations located in the ligand-binding pockets of the N- or C-terminal SH2 domains do not increase basal activity and likely exert their pathogenicity through alternative mechanisms. We lack a molecular understanding of how these SH2 mutations impact SHP2 structure, activity, and signaling. Here, we characterize five SHP2 SH2 domain ligand-binding pocket mutants through a combination of high-throughput biochemical screens, biophysical and biochemical measurements, and molecular dynamics simulations. We show that while some of these mutations alter binding affinity to phosphorylation sites, the T42A mutation in the N-SH2 domain is unique in that it also substantially alters ligand-binding specificity, despite being 8 to 10 A from the specificity-determining region of the SH2 domain. This mutation exerts its effect on sequence specificity by remodeling the phosphotyrosine-binding pocket, altering the mode of engagement of both the phosphotyrosine and surrounding residues on the ligand. The functional consequence of this altered specificity is that the T42A mutant has biased sensitivity toward a subset of activating ligands and enhances downstream signaling. Our study highlights an example of a nuanced mechanism of action for a disease-associated mutation, characterized by a change in protein--protein interaction specificity that alters enzyme activation. [ABSTRACT FROM AUTHOR]

Advancing the integration of nonliving and living components relies heavily on functional hydrogel materials with biocompatibility and customizability. In this study, an enzyme‐assisted surface activation method is developed to produce reactive hydrogel microparticles (HMPs) comprising thiolated hyaluronic acid and hyperbranched poly(β‐hydrazide esters). Fluorescence labeling analysis reveals an over six‐fold increase in surface‐active functional group density on the hydrogels and three‐fold on HMPs after enzyme activation. This enhancement improves accessibility of active elements, facilitating post‐functionalization and optimizing their capacity to support initial cell adhesion and spreading as carriers for cell cultures. Additionally, by utilizing the exposed reactive double bonds on the HMP surfaces post‐enzymatic treatment, thiolated HMPs are produced through a thiol‐ene coupling reaction with thiolated polymers. These thiolated HMPs bond together spontaneously, resulting in the formation of annealed granular hydrogels with interconnected large‐pore networks and a tunable storage modulus (G') from tens to hundreds of pascals, compatible with most soft tissues. Integrated with 3D bioprinting, this hydrogel ink generates prints that foster cell adhesion, migration, growth, and network formation. Moving forward, integrating various granular hydrogel scaffold systems with the enzyme‐assisted activation technique holds significant promise for enhancing performance and expanding applications in regenerative medicine and innovative living materials. [ABSTRACT FROM AUTHOR]

The ever-increasing uses of cellulase in various industries have made it popular among researchers. Annually, a large amount of fruit wastes go into vain, which is a great source of cellulases. The objective of this study is to use grape wastes as substrate for production of cellulases from Aspergillus niger using static fermentation. CMCase and FPase assays were performed to characterize the cellulases. The cellulases' CMCase and FPase activities and stabilities were analyzed for optimum temperature and pH. The effect of substrate concentration and kinetic constants Km and Vmax, along with thermodynamic analysis, were determined. The effects of several metals on the activity of the enzyme were observed. The optimal temperatures were found as 40 °C and 50 °C for CMCase and FPase activity, respectively. CMCase activity shows stability at 20 °C-60 °C, FPase shows low thermal stability as its activity starts to decrease after 50 °C. CMCase and FPase both show maximum activity at pH 6, and maintain their stability at pH 6-7. The values of Km and Vmax obtained from Lineweaver and Burk plot for CMCase are 0.648 mM and 12.953 mM/min, and for FPase are 0.975 mM and 41.493 mM/min. The Arrhenius plot was used to calculate activation energy (Ea) as -19 kJ/mol, and enthalpy of reaction (ΔH) as 16.4 kJ/mol, while entropy ΔS -16.4 kJ/mol was obtained from the plot of ln(Vmax/T) versus the inverse of temperature (1/T). Most metals induce enzyme activities, whereas EDTA inhibits enzyme activities. The findings suggest A. niger has remarkable cellulase production potential from grape wastes in static fermentation, at optimum temperature and pH levels for achieving enzyme activity and stability. [ABSTRACT FROM AUTHOR]

Aloe striata Haw. (AS) is one of the beneficial Aloe species that is still underutilized. In this sense, phytochemical characterization, and biological investigation of AS gel protective efficacy against paracetamol (APAP) induced hepatic injury were evaluated in a mice model, with and without loading on chitosan nanoparticles (AS gel and CS-AS NPs, respectively). Spectrophotometric determination of AS gel showed high total soluble carbohydrate content representing 3.41±0.07 μg glucose equivalent (G eq)/mg and its protein content was 0.176±0.03 g/100 g. Twenty-two metabolites [sugars (90.23 %) and organic acids (5.80 %)] were determined by GC–MS, while 7 sugars were identified and quantified by HPLC where mannose (5679.73±4.28 μg) was the predominant. Chitosan gel nanoparticles were prepared and analyzed by dynamic light scattering, high resolution transmission electron microscopy, and X-ray diffraction methods. The CS-AS NPs were the most potent antioxidant of all tested samples with respect to quercetin in several in vitro antioxidant assays. Oxidative status, architectural or immuno-histochemical and histological signs in the context of the presence of caspase-3, c-Jun-N-terminal kinase (JNK), and extracellular signal-regulated kinase 1 (ERK-1) genes were evaluated in liver AS gel could significantly restore liver biochemical, antioxidant, and anti-inflammatory activities and liver architecture. Moreover, caspase-3 contents, and ERK-1 and JNK genes expression were significantly downregulated compared to the APAP group. CS-AS NPs showed superior hepatoprotective activity than AS. CS-AS NPs hepatoprotective mechanisms could be attributed to its activation of antioxidant enzymes, and downregulation of malondialdehyde pro-inflammatory markers along with apoptosis and anti-inflammatory related genes (BAX, JNK, and ERK-1 genes). To the best of our knowledge, it is the first study reporting the hepatoprotective efficiency of AS gel and its CS-AS NPs. [Display omitted] • Quality control for multi-components in A. striata gel by GC–MS and HPLC was done. • Antioxidant and hepatoprotective properties of A. striata gel were evaluated. • The gel could preserve the normal functional status of the liver in APAP toxicity. • Hepatoprotective action was enhanced by loading the gel on chitosan nanoparticles. • Aloe striata gel and chitosan nanoparticles inhibited ERK and JNK gene expression. [ABSTRACT FROM AUTHOR]

Small synthetic oligodeoxynucleotides (ODNs) can mimic microbial nucleic acids by interacting with receptor systems and promoting immunostimulatory activities. Nevertheless, some ODNs can act differently on the plasmacytoid dendritic cell (pDC) subset, shaping their immunoregulatory properties and rendering them suitable immunotherapeutic tools in several clinical settings for treating overwhelming immune responses. We designed HIV–1–derived, DNA- and RNA-based oligonucleotides (gag, pol, and U5 regions) and assessed their activity in conferring a tolerogenic phenotype to pDCs in skin test experiments. RNA-but not DNA-oligonucleotides are capable of inducing tolerogenic features in pDCs. Interestingly, sensing the HIV–1–derived single-stranded RNA-gag oligonucleotide (RNA-gag) requires both TLR3 and TLR7 and the engagement of the TRIF adaptor molecule. Moreover, the induction of a suppressive phenotype in pDCs by RNA-gag is contingent upon the induction and activation of the immunosuppressive enzyme Arginase 1. Thus, our data suggest that sensing of the synthetic RNA-gag oligonucleotide in pDCs can induce a suppressive phenotype in pDCs, a property rendering RNA-gag a potential tool for therapeutic strategies in allergies and autoimmune diseases. [ABSTRACT FROM AUTHOR]

Plants, being sessile organisms, rely on their antioxidant systems to respond to the stress factors induced by both abiotic and biotic stresses in their environment. Among abiotic stress factors, salinity and alkalinity pose the most significant challenges to plant growth. To counteract these stresses, plants activate various signalling pathways to enhance their stress tolerance. While a wide range of pesticides, including insecticides and herbicides, are employed to protect agricultural crops from biotic agents, there exists no established practice for fortifying their defence mechanisms against abiotic stresses. This study delves into the effect of H2O2 pre-treatment on mitigating salt stress in tomato seedlings. Four experimental groups were established: control, H2O2, Salt, and Salt+H2O2. The study evaluated changes in chlorophyll content, malondialdehyde (MDA) accumulation, superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) enzyme activities and expressions. The results revealed that priming treatment led to increased chlorophyll levels and reduced MDA accumulation compared to the group only exposed to salt stress. Additionally, the activation of stress-related enzymes was significantly higher in the priming group compared to the group only exposed to salt stress. Expression levels exhibited a statistically significant increase compared to the control group; however, CAT and APX expression levels were found to be lower than those in the the group only exposed to salt stress. These findings suggest that H2O2 priming can enhance plant stress tolerance. Priming can serve as a highly effective tool to alleviate stress in plants; however, the type, concentration, and exposure time of the priming agent are crucial factors in regulating the priming effect. [ABSTRACT FROM AUTHOR]

To cope with a high-salinity environment, haloarchaea generally employ the twin-arginine translocation (Tat) pathway to transport secretory proteins across the cytoplasm membrane in a folded state, including Tat-dependent extracellular subtilases (halolysins) capable of autocatalytic activation. Some halolysins, such as SptA of Natrinema gari J7-2, are produced at late-log phase to prevent premature enzyme activation and proteolytic damage of cellular proteins in haloarchaea; however, the regulation mechanism for growth phase-dependent expression of halolysins remains largely unknown. In this study, a DNA-protein pull-down assay was performed to identify the proteins binding to the 5'-flanking sequence of sptA encoding halolysin SptA in strain J7-2, revealing a TrmBL2-like transcription factor (NgTrmBL2). The ΔtrmBL2 mutant of strain J7-2 showed a sharp decrease in the production of SptA, suggesting that NgTrmBL2 positively regulates sptA expression. The purified recombinant NgTrmBL2 mainly existed as a dimer although monomeric and higher-order oligomeric forms were detected by native-PAGE analysis. The results of electrophoretic mobility shift assays (EMSAs) showed that NgTrmBL2 binds to the 5'-flanking sequence of sptA in a nonspecific and concentration-dependent manner and exhibits an increased DNA-binding affinity with the increase in KCl concentration. Moreover, we found that a distal cisregulatory element embedded in the neighboring upstream gene negatively regulates trmBL2 expression and thus participates in the growth phase-dependent biosynthesis of halolysin SptA. [ABSTRACT FROM AUTHOR]

Salinity poses a significant threat to agricultural productivity worldwide, with its detrimental effects on plant growth and physiological processes. Understanding the mechanisms by which plants respond to salt stress is crucial for developing strategies to mitigate its impact on crop yield and sustainability. To address this issue, a pot study was conducted to determine the effect of salt stress on the physiological and biochemical attributes of neem (Azdiarchta indica A. Juss). Plants were grown for 10 months in pots filled with soil having different salinity levels of 3, 6, 9, 12, 15, and 18 dS m−1 and compared with a control of 1.7 dS m−1. The results showed that plant growth and chlorophyll contents declined as salinity levels increased. Due to oxidative stress, the contents of H2O2 increased under higher salt levels. The mitigation of oxidative stress was achieved through the activation of antioxidant enzymes (catalase, ascorbate peroxidase, peroxidase, and superoxide dismutase). Multivariate analysis indicated that Na+ accumulation in plants was positively related to H2O2 production and enzymatic activities, and negatively related to plant biomass, chlorophyll contents, root and shoot K+ concentration, and root K+/Na+. The experimental results suggest that neem plants can be grown in moderate saline soils. [ABSTRACT FROM AUTHOR]

Postharvest avocado is susceptible to attack by Neofusicoccum parvum, which has been reported to cause black spot in avocado pulp. Therefore, it is necessary to look for alternatives for its control with products that are low-cost, effective and without risks to human health and the environment, and that also stimulate the defense mechanisms of the fruit. The aim was to evaluate the effect of basic and GRAS substance treatments on the in vitro control of N. parvum, and the induction of enzymes related to the defense mechanisms of the fruit. N. parvum was isolated from avocado fruit. Morphological and molecular identification was performed. In vitro and in vivo treatments were made for the control of pathogens and the induction of defense mechanisms in the fruit with basic and GRAS substance treatments based on chitosan and GRAS substances. The basic and GRAS substance treatments inhibited the development of N. parvum mycelium by 80–100%. In addition, they induced the activation of enzymes related to the defense mechanisms of the fruit (PAL, POD and PPO). The best basic and GRAS substance treatments, both in vitro and in vivo, were those based on chitosan (0.5%) and cinnamon essential oil (2.5%) when they were applied combined in the form of an emulsion. [ABSTRACT FROM AUTHOR]

Ufmylation is implicated in multiple cellular processes, but little is known about its functions and regulation in protein trafficking. Here, we demonstrate that the genetic depletion of core components of the ufmylation cascade, including ubiquitin-fold modifier 1 (UFM1), UFM1 activation enzyme 5, UFM1-specific ligase 1 (UFL1), UFM1- specific protease 2, and UFM1-binding protein 1 (UFBP1) each markedly inhibits the endoplasmic reticulum (ER)-Golgi transport, surface delivery, and recruitment to COPII vesicles of a subset of G protein-coupled receptors (GPCRs) and UFBP1's function partially relies on UFM1 conjugation. We also show that UFBP1 and UFL1 interact with GPCRs and UFBP1 localizes at COPII vesicles coated with specific Sec24 isoforms. Furthermore, the UFBP1/UFL1-binding domain identified in the receptors effectively converts non-GPCR protein transport into the ufmylation-dependent pathway. Collectively, these data reveal important functions for the ufmylation system in GPCR recruitment to COPII vesicles, biosynthetic transport, and sorting at ER via UFBP1 ufmylation and interaction directly. [ABSTRACT FROM AUTHOR]

Imaging of mitophagy is of significance as aberrant mitophagy is engaged in multiple diseases. Mitophagy has been imaged with synthetic or biotic pH sensors by reporting pH acidification en route delivery into lysosomes. To circumvent uncertainty of acidity-dependent signals, we herein report an enzyme-activatable probe covalently attached on mitochondrial inner membrane (ECAM) for signal-persist mitophagy imaging. ECAM is operated via ΔΨm-driven accumulation of Mito-proGreen in mitochondria and covalent linking of the trapped probe with azidophospholipids metabolically incorporated into the mitochondrial inner membrane. Upon mitophagy, ECAM is delivered into lysosomes and hydrolyzed by LNPEP/leucyl aminopeptidase, yielding turn-on green fluorescence that is immune to lysosomal acidity changes and stably retained in fixed cells. With ECAM, phorbol-12-myristate-13-acetate (PMA) was identified as a highly potent inducer of mitophagy. Overcoming signal susceptibility of pH probes and liability of ΔΨm probes to dissipation from stressed mitochondria, ECAM offers an attractive tool to study mitophagy and mitophagy-inducing therapeutic agents.Abbreviations: Baf-A1, bafilomycin A1; CCCP, carbonyl cyanide m-chlorophenylhydrazone; DBCO, dibenzocyclooctyne; ECAM, enzyme-activated probe covalently attached on mitochondrial inner membrane; GFP, green fluorescent protein; LAMP2, lysosomal associated membrane protein 2; LNPEP/LAP, leucyl and cystinyl aminopeptidase; PMA, phorbol-12-myristate-13-acetate; ΔΨm, mitochondrial transmembrane potential; RFP, red fluorescent protein; TPP, triphenylphosphonium. [ABSTRACT FROM AUTHOR]

Background: Peanut (Arachis hypogaea), a vital oil and food crop globally, is susceptible to web blotch which is a significant foliar disease caused by Phoma arachidicola Marasas Pauer&Boerema leading to substantial yield losses in peanut production. Calcium treatment has been found to enhance plant resistance against pathogens. Results: This study investigates the impact of exogenous calcium on peanut resistance to web blotch and explores its mechanisms. Greenhouse experiments revealed that exogenous calcium treatment effectively enhanced resistance to peanut web blotch. Specifically, amino acid calcium and sugar alcohol calcium solutions demonstrated the best induced resistance effects, achieving reduction rates of 61.54% and 60% in Baisha1016, and 53.94% and 50% in Luhua11, respectively. All exogenous calcium treatments reduced malondialdehyde (MDA) and relative electrical conductivity (REC) levels in peanut leaves, mitigating pathogen-induced cell membrane damage. Exogenous calcium supplementation led to elevated hydrogen peroxide (H2O2) content and superoxide anion (O2∙-) production in peanut leaves, facilitating the accumulation of reactive oxygen species (ROS) crucial for plant defense responses. Amino acid calcium and sugar alcohol calcium treatments significantly boosted activities of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) in peanut leaves. Activation of these antioxidant enzymes effectively scavenged excess ROS, maintaining ROS balance and mitigating cellular damage. Conclusions: In summary, exogenous calcium treatment triggered ROS production, which was subsequently eliminated by the activation of antioxidant enzymes, thereby reducing cell membrane damage and inducing defense responses against peanut web blotch. [ABSTRACT FROM AUTHOR]

Enzymatic hydrolysis is a green approach for nutrient bioconversion of sea cucumber body wall to recover sulfated polysaccharides. However, formation of high level of advanced glycation end products (AGEs) can potentially negate health benefits of the functional food. In this study, UV-assisted autolysis was employed to optimize nutrient bioconversion of Stichopus horrens body wall using extreme UV dosage (324,000-648,000 J/m2), different autolysis temperatures (40–60 °C) and time (3–5 h). Parallel biochemical changes indicate that UV irradiation promoted the activation of endogenous enzymes, leading to the proteolysis of the body wall and the subsequent dissolution of sulfated polysaccharides. Changes in the amide band I region of the Fourier-transform infrared (FTIR) spectra of autolyzed body wall residue reflected the loss of α-helix secondary structures of collagen which exacerbated at autolysis temperatures beyond 50 °C. The maximum yield of sulfated polysaccharides and antioxidant activities were obtained at the condition (486,000 J/m2 of UV dosage, 60 ℃, and 4 h of autolysis) by the optimization using the central composite design. The nutritional composition and safety aspect of the autolysate was compared with hydrolysate from enzymatic hydrolysis and hot water extract. Quantification of 5-hydroxymethylfurfural (HMF) showed that UV-assisted autolysis could mitigate HMF formation (0.12 mg/kg) by 97.6% compared to enzymatic hydrolysis (5.13 mg/kg) and 42.9% compared to hot water extraction (0.21 mg/kg). The UV-assisted autolysis with metal ion further enhanced the extraction process of sulfated polysaccharides from sea cucumber body wall and reduction of the processing contaminants. Thus, UV-assisted autolysis offers a safer alternative for the nutrient bioconversion of sea cucumber body wall, particularly for the development of functional foods. [ABSTRACT FROM AUTHOR]

Chirality plays a significant role in the manufacture of pharmaceuticals and fine chemicals. The use of chemical catalysts to control stereoselectivity relies on the use of chiral catalysts with labor–intensive synthesis and purification. Natural enzymes offer inherent stereoselectivity, making them attractive catalysts for this purpose. We report here chiral biocatalytic oxidations in microemulsions driven by horseradish peroxidase coupled with a synthetic Cu2+‐polymer catalyst. This hybrid system features crosslinked layer–by–layer (LBL) films composed of polyions with Cu2+‐containing pyrene–labelled poly(2‐hydroxy‐3‐dipicolylamino) propyl methacrylate (Py−PGMADPA) to drive oxygen reduction to form hydrogen peroxide. Peroxide in turn activates horseradish peroxidase (HRP) crosslinked in LbL films on magnetic particle beads to biocatalytically oxidize styrene, ethylbenzene, and methyl phenylacetate to chiral products. R‐stereoisomers of these reactants were selectively formed with a high enantiomeric excess of ≥80 % at 90 °C. The enzyme films show high thermal stability at 90 °C in cetyltrimethylammonium bromide microemulsion. Reactions at 90 °C were essentially complete in 2 hr. This hybrid approach opens a door to new designs of biocatalytic syntheses using a separate electrocatalyst for enzyme activation. [ABSTRACT FROM AUTHOR]

Acaricides used against Tetranychus urticae Koch, 1836 (Acari: Tetranychidae) in cotton fields cause control failure over time. To determine the resistance status of T. urticae populations to tebufenpyrad and bifenazate, different populations collected from Aydın (AYD), Adana (ADA), Şanlıurfa (SAN), and Diyarbakır (DIY) provinces of Türkiye, between 2019 and 2020, were subjected to diagnostic dose bioassays. Firstly, the spider mites were eliminated with a discriminating dose. Afterwards, LC50 and LC90 of the remaining populations were determined and the ten highest resistant populations were selected. The highest phenotypic resistance to bifenazate was observed in AYD4 and DIY2 (LC50 57.14 mg L− 1 with 85.01-fold and LC50 30.15 mg L− 1with 44.86-fold, respectively), while the lowest phenotypic resistance was found in SAN6 (LC50 1.5 mg L− 1; 2.28-fold). Considering the phenotypic resistance to tebufenpyrad, the highest resistance was found in AYD4 population (LC50 96.81 mg L− 1; 12.92-fold), while the lowest - in DIY28 population (LC50 21.23 mg L− 1; 2.83-fold). In pharmacokinetic studies, the ADA16 population was compared with the sensitive German Susceptible Strain population and it was determined that carboxylesterase activity was statistically higher (1.46 ± 0.04 nmol/min/mg protein enzyme activation 2.70-fold). The highest activation of glutathione S-transferase was detected in ADA16 (1.49 ± 0.01 nmol/min/mg protein; 2.32-fold). No mutations were found in PSST (METI 1), the point mutation site for tebufenpyrad, and Cytb (METI 3), the point mutation site for bifenazate. In terms of phenotypic resistance, bifenazate was found to be moderately resistant in two populations (85.01 and 44.86-fold), while tebufenpyrad was moderately resistant in one population (12.92-fold). This study showed that both acaricides are still effective against T. urticae populations. [ABSTRACT FROM AUTHOR]

Activation of the CRISPR‐Cas13a system requires the formation of a crRNA‐Cas13a ribonucleoprotein (RNP) complex and the binding of an RNA activator to the RNP. These two binding processes play a crucial role in the performance of the CRISPR‐Cas13a system. However, the binding kinetics remain poorly understood, and a main challenge is the lack of a sensitive method for real‐time measurements of the dynamically formed active CRISPR‐Cas13a enzyme. We describe here a new method to study the binding kinetics and report the rate constants (kon and koff) and dissociation constant (Kd) for the binding between Cas13a and its activator. The method is able to unravel and quantify the kinetics of binding and cleavage separately, on the basis of measuring the real‐time trans‐cleavage rates of the CRISPR‐Cas system and obtaining the real‐time concentrations of the active CRISPR‐Cas ternary complex. We further discovered that once activated, the Cas13a system operates at a wide range of temperatures (7–37 °C) with fast trans‐cleavage kinetics. The new method and findings are important for diverse applications of the Cas13a system, such as the demonstrated quantification of microRNA at ambient temperatures (e.g. 25 °C). [ABSTRACT FROM AUTHOR]

Severe acute pancreatitis (SAP) begins with premature activation of enzymes, promoted by the immune system, triggering a potential systemic inflammatory response that leads to organ failure with increased mortality and a bleak prognosis. Interleukin-22 (IL-22) is a cytokine that may have a significant role in SAP. IL-22, a member of the IL-10 cytokine family, has garnered growing interest owing to its potential tissue-protective properties. Recently, emerging research has revealed its specific effects on pancreatic diseases, particularly SAP. This paper provides a review of the latest knowledge on the role of IL-22 and its viability as a therapeutic target in SAP. Highlights: 1. Interleukin-22 (IL-22) is a member of the IL-10 family of cytokines. 2. IL-22 is a potential indicator for monitoring organ dysfunction and severity of severe acute pancreatitis (SAP). 3. IL-22 acts as a protective factor against acinar cell damage by suppressing autophagy and reducing the infiltration of inflammatory cells. 4. IL-22 alleviate SAP-induced multiorgan injury. 5. IL-22 protect SAP-induced intestinal barrier damage. [ABSTRACT FROM AUTHOR]

T cells are crucial for adaptive immunity to regulate proper immune response and immune homeostasis. T cell development occurs in the thymus and mainly differentiates into CD4+ and CD8+ T cell subsets. Upon stimulation, naive T cells differentiate into distinct CD4+ helper and CD8+ cytotoxic T cells, which mediate immunity homeostasis and defend against pathogens or tumours. Trace elements are minimal yet essential components of human body that cannot be overlooked, and they participate in enzyme activation, DNA synthesis, antioxidant defence, hormone production, etc. Moreover, trace elements are particularly involved in immune regulations. Here, we have summarized the roles of eight essential trace elements (iron, zinc, selenium, copper, iodine, chromium, molybdenum, cobalt) in T cell development, activation and differentiation, and immune response, which provides significant insights into developing novel approaches to modulate immunoregulation and immunotherapy. [ABSTRACT FROM AUTHOR]

Until recently, the main pharmaceuticals used to control cholesterol and prevent cardiovascular disease (CVD) were statin-related drugs, known for their historical side effects. Therefore, there is growing interest in exploring alternatives, such as nutritional and dietary components, that could play a central role in CVD prevention. This review aims to provide a comprehensive understanding of how natural phytosterols found in various diets combat CVDs. We begin with a description of the overall approach, then we explore in detail the different direct and indirect mechanisms that contribute to reducing cardiovascular incidents. Phytosterols, including stigmasterol, β-sitosterol, ergosterol, and fucosterol, emerge as promising molecules within nutritional systems for protection against CVDs due to their beneficial effects at different levels through direct or indirect cellular, subcellular, and molecular mechanisms. Specifically, the mentioned phytosterols exhibit the ability to diminish the generation of various radicals, including hydroperoxides and hydrogen peroxide. They also promote the activation of antioxidant enzymes such as superoxide dismutase, catalase, and glutathione, while inhibiting lipid peroxidation through the activation of Nrf2 and Nrf2/heme oxygenase-1 (HO-1) signaling pathways. Additionally, they demonstrate a significant inhibitory capacity in the generation of pro-inflammatory cytokines, thus playing a crucial role in regulating the inflammatory/immune response by inhibiting the expression of proteins involved in cellular signaling pathways such as JAK3/STAT3 and NF-κB. Moreover, phytosterols play a key role in reducing cholesterol absorption and improving the lipid profile. These compounds can be used as dietary supplements or included in specific diets to aid control cholesterol levels, particularly in individuals suffering from hypercholesterolemia. [ABSTRACT FROM AUTHOR]

Photodynamic therapy (PDT) emerges as a novel approach to treat tumors utilizing "photosensitizers" and "light". This method involves exposing the tumor site to specific wavelengths of light, activating photosensitizers concentrated in the tumor to generate highly oxidizing reactive oxygen species, ultimately dismantling the tumor. In comparison to surgery, chemotherapy, and radiation therapy, PDT boasts simplicity in operation, precise targeting, minimal invasiveness, lack of drug resistance, and the ability to trigger systemic immunity. While PDT has gained widespread adoption in developed nations, its promotion in China is still underway, hampered by a lack of public understanding. In response, we present a popular science experiment elucidating tumor-targeted photodynamic therapy using a laboratory-synthesized selenium-Rhodamine photosensitizer. The experiment encompasses enzyme activation of targeted photosensitizers, visual detection of singlet oxygen, and the photodynamic eradication of cancer cells. This experiment vividly illustrates the scientific principles behind targeted photodynamic therapy, enhancing public awareness of photodynamic cancer treatment. Such efforts are crucial for advancing the adoption of photodynamic therapy in our country, underscoring the pivotal role of chemistry in cancer treatment. The entire popular science experiment is straightforward, visually intuitive, and has demonstrated effective results in science outreach. [ABSTRACT FROM AUTHOR]

Cotton is an important plant-based protein. Cottonseed cake, a byproduct of the biodiesel industry, offers potential in animal supplementation, although the presence of the antinutritional sesquiterpenoid gossypol limits utilization. The macrofungus Panus lecomtei offers potential in detoxification of antinutritional factors. Through an enzymatic and proteomic analysis of P. lecomtei strain BRM044603, grown on crushed whole cottonseed contrasting in the presence of free gossypol (FG), this study investigated FG biodegradation over a 15-day cultivation period. Fungal growth reduced FG to levels at 100 μg/g, with a complex adaptive response observed, involving primary metabolism and activation of oxidative enzymes for metabolism of xenobiotics. Increasing activity of secreted laccases correlated with a reduction in FG, with enzyme fractions degrading synthetic gossypol to trace levels. A total of 143 and 49 differentially abundant proteins were observed across the two contrasting growth conditions after 6 and 12 days of cultivation, respectively, revealing a dynamic protein profile during FG degradation, initially related to constitutive metabolism, then later associated with responses to oxidative stress. The findings advance our understanding of the mechanisms involved in gossypol degradation and highlight the potential of P. lecomtei BRM044603 in cotton waste biotreatment, relevant for animal supplementation, sustainable resource utilization, and bioremediation. [ABSTRACT FROM AUTHOR]

Serratia marcescens is an emerging health‐threatening, gram‐negative opportunistic pathogen associated with a wide variety of localized and life‐threatening systemic infections. One of the most crucial virulence factors produced by S. marcescens is serratiopeptidase, a 50.2‐kDa repeats‐in‐toxin (RTX) family broad‐specificity zinc metalloprotease. RTX family proteins are functionally diverse exoproteins of gram‐negative bacteria that exhibit calcium‐dependent structural dynamicity and are secreted through a common type‐1 secretion system (T1SS) machinery. To evaluate the impact of various divalent ligands on the folding and maturation of serratiopeptidase zymogen, the protein was purified and a series of structural and functional investigations were undertaken. The results indicate that calcium binding to the C‐terminal RTX domain acts as a folding switch, triggering a disordered‐to‐ordered transition in the enzyme's conformation. Further, the auto‐processing of the 16‐amino acid N‐terminal pro‐peptide results in the maturation of the enzyme. The binding of calcium ions to serratiopeptidase causes a highly cooperative conformational transition in its structure, which is essential for the enzyme's activation and maturation. This conformational change is accompanied by an increase in solubility and enzymatic activity. For efficient secretion and to minimize intracellular toxicity, the enzyme needs to be in an unfolded extended form. The calcium‐rich extracellular environment favors the folding and processing of zymogen into mature serratiopeptidase, i.e., the holo‐form required by S. marcescens to establish infections and survive in different environmental niches. [ABSTRACT FROM AUTHOR]

Cyclic nucleotides are the most diversified category of second messengers and are found in all organisms modulating diverse pathways. While cAMP and cGMP have been studied over 50 years, cyclic di‐nucleotide signaling in eukaryotes emerged only recently with the anti‐viral molecule 2´3´cGAMP. Recent breakthrough discoveries have revealed not only the astonishing chemical diversity of cyclic nucleotides but also surprisingly deep‐rooted evolutionary origins of cyclic oligo‐nucleotide signaling pathways and structural conservation of the proteins involved in their synthesis and signaling. Here we discuss how enzyme‐centered approaches have paved the way for the identification of several cyclic nucleotide signals, focusing on the advantages and challenges associated with deciphering the activation mechanisms of such enzymes. [ABSTRACT FROM AUTHOR]

Emerging consumer demand for safer, more sustainable flavors and fragrances has created new challenges for the industry. Enzymatic syntheses represent a promising green production route, but the broad application requires engineering advancements for expanded diversity, improved selectivity, and enhanced stability to be cost-competitive with current methods. This review discusses recent advances and future outlooks for enzyme engineering in this field. We focus on carboxylic acid reductases (CARs) and unspecific peroxygenases (UPOs) that enable selective productions of complex flavor and fragrance molecules. Both enzyme types consist of natural variants with attractive characteristics for biocatalytic applications. Applying protein engineering methods, including rational design and directed evolution in concert with computational modeling, present excellent examples for property improvements to unleash the full potential of enzymes in the biosynthesis of value-added chemicals. • Carboxylic acid reductases (CARs) directly reduce carboxylic acids into aldehydes without a second enzyme for substrate activation. • Unspecific peroxygenases (UPOs) are self-sufficient mono peroxygenases that catalyze similar reactions as P450 enzymes. • Both groups of enzymes have broad substrate scopes and perform chemistries that are challenging to chemical methods. • We present enzyme engineering examples that improved and/or altered the catalytic property or stability of these enzymes. • The engineering achievements are steppingstones towards the ultimate industrial syntheses using CARs and UPOs. [ABSTRACT FROM AUTHOR]

Ultrasound has attracted attention in recent years as a green and effective non-thermal processing technique used in various fields, especially in the food, biotechnology and biopharmaceutical industries. It is important to determine the effects of ultrasonication on enzymatic reactions that have important applications in these industries. While intense ultrasonication conditions cause enzyme inactivation, it has been reported that enzyme activity can be increased under mid-ultrasound conditions (i.e. low intensity and short duration). Ultrasound treatment can be applied to free enzyme, substrate and immobilized enzyme. Ultrasonication may have a positive effect on the structure of molecules, increasing enzyme activity and product yield. In this review, information is given about the changes in molecular structure and enzyme activation that occur as a result of the application of ultrasound treatment to free enzyme, immobilized enzyme and substrate, the mechanisms of these changes and the factors that affect these mechanisms. [ABSTRACT FROM AUTHOR]

We explored the impact of strontium oxide nanoparticles (SrO-NPs), synthesized through a green method, on seedling growth of bread wheat in hydroponic systems. The wheat plants were exposed to SrO-NPs concentrations ranging from 0.5 mM to 8.0 mM. Various parameters, including shoot length (cm), shoot fresh weight (g), root number, root length (cm), root fresh weight (g), chlorophyll value (SPAD), cell membrane damage (%), hydrogen peroxide (H2O2) value (µmol/g), malondialdehyde (MDA) value (ng/µL), and enzymatic activities like ascorbate peroxidase (APX) activity (EU/g FW), peroxidase (POD) activity (EU/g FW), and superoxide dismutase (SOD) activity (U/g FW), were measured to assess the effects of SrO-NPs on the wheat plants in hydroponic conditions. The results showed that the SrO-NPs in different concentrations were significantly affected considering all traits. The highest values were obtained from the shoot length (20.77 cm; 0.5 mM), shoot fresh weight (0.184 g; 1 mM), root number (5.39; 8 mM), root length (19.69 cm; 0 mM), root fresh weight (0.142 g; 1 mM), SPAD (33.20; 4 mM), cell membrane damage (58.86%; 4 mM), H2O2 (829.95 µmol/g; 6 mM), MDA (0.66 ng/µl; 8 mM), APX (3.83 U/g FW; 6 mM), POD (70.27 U/g FW; 1.50 mM), and SOD (60.77 U/g FW; 8 mM). The data unequivocally supports the effectiveness of SrO-NPs application in promoting shoot and root development, chlorophyll levels, cellular tolerance, and the activation of enzymes in wheat plants. [ABSTRACT FROM AUTHOR]

Summary: Barley has played a critical role in mankind civilisation. Despite being underexploited, highland barley (HB) is an economical crop demonstrating superior nutritional and health benefits compared to most common cereals. In recent years, germination has drawn a great attention because of its ability to increase nutrients in grains. This review aimed to elucidate the implication of germination in improving the qualities of HB. Because of germination, amelioration in nutrients and digestibility was observed in germinated HB (GHB) than ungerminated HB, due to activation of various enzymes. An increase in polyphenols and antioxidant activity was achieved in GHB. GHB‐based products exhibited better water‐holding capacity, water solubility index, oil‐holding capacity, sensory attributes and cooking time. However, excessive germination may have negative impact on starch‐protein network, which might be detrimental during food processing. Overall, controlled‐germination could be a viable approach to improve the digestibility, palatability, acceptability and shelf‐life of HB‐based products. [ABSTRACT FROM AUTHOR]

An endophytic symbiont P. aeruginosa-producing anticandidal siderophore was recovered from mangrove leaves for the first time. Production was optimal in a succinate medium supplemented with 0.4% citric acid and 15 µM iron at pH 7 and 35 °C after 60 h of fermentation. UV spectra of the acidic preparation after purification with Amberlite XAD-4 resin gave a peak at 400 nm, while the neutralized form gave a peak at 360 nm. A prominent peak with RP-HPLC was obtained at RT 18.95 min, confirming its homogeneity. It was pH stable at 5.0–9.5 and thermally stable at elevated temperatures, which encourages the possibility of its application in extreme environments. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) against Candida spp. Were in the range of 128 µg/mL and lower. It enhanced the intracellular iron accumulation with 3.2–4.2-fold (as judged by atomic absorption spectrometry) with a subsequent increase in the intracellular antioxidative enzymes SOD and CAT. Furthermore, the malondialdehyde (MDA) concentration due to cellular lipid peroxidation increased to 3.8-fold and 7.3-fold in C. albicans and C. tropicalis, respectively. The scanning electron microscope (SEM) confirmed cellular damage in the form of roughness, malformation, and production of defensive exopolysaccharides and/or proteins after exposure to siderophore. In conclusion, this anticandidal siderophore may be a promising biocontrol, nonpolluting agent against waterborne pathogens and pathogens of the skin. It indirectly kills Candida spp. by ferroptosis and mediation of hyperaccumulation of iron rather than directly attacking the cell targets, which triggers the activation of antioxidative enzymes. [ABSTRACT FROM AUTHOR]